Exposure of the myocardium to moderate levels (10mM) of ethanol (ETOH)appears to exert a beneficial protective effect against subsequent myocardial ischemia. Further clinical trials have also shown that patients having suffered from an ischemic event, benefit from moderate ETOH ingestion. However, the molecular mechanism(s) through which this protection occur(s) is not known. This proposal will further develop a model adult cardiomyocyte culture system which closely mimics the 3-D contracting in situ state of the intact adult myocardium and to employ this system to investigate the alterations in the adhesive portions of the cytoskeleton following exposure of the cardiomyocytes to graded (0 yields 10mM) ETOH. Our investigation will focus on costameres, a complex of numerous cytoskeletal proteins which link the myofibrillar contractile mechanism to the extracellular matrix thereby transmitting the generated systolic force to the surrounding matrix which is complex in heart. The use of a newly developed fluorescent probe, green fluorescent protein (GFP) which can be synthesized within the cardiomyocytes will permit us to follow changes in this cytoskeletal complex in real time using our DeltaVision deconvolution microscope/computer system employing rapid acquistion, 3-D reconstruction of two specific proteins, tensin and vinculin which we have observed to be modified during ETOH exposure. We shall also examine potential changes in the activity and specificity of a protein kinase C isoform (epsilon), a phosphorylating enzyme localized in the costameric regions which possibly may act as a catalyst regulating the cytoskeletal structural changes. It is our hypothesis that alterations in this region of the cytoskeleton adhesion system reduce stress on the contractile apparatus thereby conserving energy (ATP) needed to maintain contractily in a compromised state such as myocardial ischemia.